RESEARCH ARTICLE
Molecular identification of two Culex (Culex) species of the neotropical region (Diptera: Culicidae) Magdalena Laurito1☯*, Ana M. Ayala2☯, Walter R. Almiro´n1‡, Cristina N. Gardenal2‡ 1 Instituto de Investigaciones Biolo´gicas y Tecnolo´gicas (IIByT) CONICET-UNC. Centro de Investigaciones Entomolo´gicas de Co´rdoba (CIEC)—Universidad Nacional de Co´rdoba, Co´rdoba, Argentina, 2 Instituto de Diversidad y Ecologı´a Animal (IDEA) CONICET -Universidad Nacional de Co´rdoba, Co´rdoba, Argentina ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * [email protected]
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OPEN ACCESS Citation: Laurito M, Ayala AM, Almiro´n WR, Gardenal CN (2017) Molecular identification of two Culex (Culex) species of the neotropical region (Diptera: Culicidae). PLoS ONE 12(2): e0173052. doi:10.1371/journal.pone.0173052 Editor: Bi-Song Yue, Sichuan University, CHINA Received: December 13, 2016 Accepted: February 14, 2017 Published: February 24, 2017 Copyright: © 2017 Laurito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All sequences have been submitted to GenBank and the accession numbers are within the paper.
Abstract Culex bidens and C. interfor, implicated in arbovirus transmission in Argentina, are sister species, only distinguishable by feature of the male genitalia; however, intermediate specimens of the species in sympatry have been found. Fourth-instar larvae and females of both species share apomorphic features, and this lack of clear distinction creates problems for specific identification. Geometric morphometric traits of these life stages also do not distinguish the species. The aim of the present study was to assess the taxonomic status of C. bidens and C. interfor using two mitochondrial genes and to determine the degree of their reproductive isolation using microsatellite loci. Sequences of the ND4 and COI genes were concatenated in a matrix of 993 nucleotides and used for phylogenetic and distance analyses. Bayesian and maximum parsimony inferences showed a well resolved and supported topology, enclosing sequences of individuals of C. bidens (0.83 BPP, 73 BSV) and C. interfor (0.98 BPP, 97 BSV) in a strong sister relationship. The mean K2P distance within C. bidens and C. interfor was 0.3% and 0.2%, respectively, and the interspecific variation was 2.3%. Bayesian clustering also showed two distinct mitochondrial lineages. All sequenced mosquitoes were successfully identified in accordance with the best close match algorithm. The low genetic distance values obtained indicate that the species diverged quite recently. Most morphologically intermediate specimens of C. bidens from Co´rdoba were heterozygous for the microsatellite locus GT51; the significant heterozygote excess observed suggests incomplete reproductive isolation. However, C. bidens and C. interfor should be considered good species: the ventral arm of the phallosome of the male genitalia and the ND4 and COI sequences are diagnostic characters.
Funding: PICT 2013-1254 http://www.agencia. mincyt.gob.ar/ Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica. ML.
Introduction
Competing interests: The authors have declared that no competing interests exist.
The genus Culex L., with a worldwide distribution and 770 species grouped in 26 subgenera [1], is widely known for its medical and veterinary importance. Among the subgenera,
PLOS ONE | DOI:10.1371/journal.pone.0173052 February 24, 2017
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Mosquito molecular identification
subgenus Culex includes 200 species [1], many of them recognized as vectors of filarial nematodes [2] and several arboviruses [3]. The subgenus is not monophyletic [4,5,6] and the infrasubgeneric categories are based on superficial similarities that may not reflect natural relationships [7]. The subgenus exhibits polymorphic features and exceptional forms [8] and is arranged in an informal classification that includes Series, Groups, Subgroups and Complexes, with nine species without placement [1]. Morphological identification of Culex (Culex) species is difficult because anatomical traits of fourth-instar larvae and females are polymorphic and overlap among species. Only morphological features of the male genitalia provide a wealth of taxonomic characters that allow reliable identification of the species, as well as synapomorphic traits that contribute to revolving phylogenetic relationships in the subgenus [9]. Culex bidens Dyar and C. interfor Dyar are a clear example of the problematic identification described above. The former is widely distributed in many countries of the Neotropical Region from Mexico to Argentina; the latter is restricted to Argentina and Paraguay [10]. The species are grouped in a well-supported clade as sister species on the basis of morphological characters [9]. These species share apomorphic features of fourth-instar larvae and females, being only distinguishable by some male genitalia traits [9]. The two species share features of the subapical lobe of the gonocoxopodites, paraproct and dorsal arms of the phallosome. Culex bidens differs by having lateral plate with 1–3 (usually 2) large dorsolaterally directed teeth, 0–3 minute conical denticles and the ventral arm developed as a spine bent dorsolaterally. The lateral plate of C. interfor has a single strong tooth directed dorsolaterally (infrequently with denticles), and the ventral arm is small, triangular and laterally directed flap-like process [11]. Besides morphological characters, geometric morphometric procedures were also assessed to distinguish these species from each other and from other species [12], revealing that shapes of wings of adults and dorsomentum of larvae could not distinguish them. Furthermore, intermediate features in male genitalia in sympatric populations of the two species have recently been found, suggesting that successful interbreeding could be occurring in the localities of Resistencia (Chaco Province), Co´rdoba and Rı´o Seco (Co´rdoba Province), Puerto Iguazu´ (Misiones Province) and Chamical (La Rioja Province) of Argentina [13]. Distinguishing closely related species using morphological traits is not easy, even more so if there are intermediate specimens. Accurate identification is of paramount importance to determine areas of potential risk of disease transmission, particularly when dealing with invasive mosquito species that are potential disease vectors. The need to distinguish C. bidens and C. interfor is important because they have been implicated in the transmission of two arboviruses. Culex bidens was assumed to be the vector of Venezuelan Equine Encephalitis virus (VEEV) during the epizootic in Chaco Province in 1988 [14] and C. interfor is considered a secondary vector of Saint Louis Encephalitis virus (SLEV) [15] in Co´rdoba, Argentina. To achieve a correct identification of these species, alternative tools, such as molecular methods, are needed. To be effective, a diagnostic molecular marker must demonstrate consistent differences between closely related species [16]. Mitochondrial genes are considered good markers because of their abundance, lack of introns, limited exposure to recombination, and haploid mode of inheritance [17]. Among mitochondrial genes, a fragment of the cytochrome c oxidase subunit I (COI) has been largely employed for taxon barcoding [18], and to estimate genetic divergence among phylogenetically close species [19]. In mosquitoes, COI barcode sequences have been used to successfully identify species [20,21,22] and to recognize species complexes [23,24,25]. Even though Laurito et al. [26] revealed that the COI barcode fragment does not contain enough information to identify all species of Culex (Culex) from the Neotropical Region, about 40% of the samples clustered with their conspecifics in that study. Another DNA region, the nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4) has
PLOS ONE | DOI:10.1371/journal.pone.0173052 February 24, 2017
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Mosquito molecular identification
recently been employed for specific identification in Aedes koreicus (Edwards) populations far away from its known natural range of distribution [27]. Cameron et al. [28] designed a molecular assay based on ND4 sequences to identify species of Aedes Meigen. The lack of appropriate diagnostic morphological characters to identify C. bidens and C. interfor in all stages except males, their close phylogenetic relationship, the sympatric occurrence of intermediate specimens and the poor differentiation based on geometric morphometrics lead us to question the taxonomic rank of these species. The aim of the present study was to assess the taxonomic status of C. bidens and C. interfor using a mitochondrial DNAbased phylogenetic molecular approach. Additionally, three microsatellite loci were tested to get complementary information based on other than maternally inherited markers, which could provide evidence about the degree of reproductive isolation between these species.
Material and methods Mosquito collection Fourth-instar larvae were collected between 2012 and 2015 in the Catamarca (28˚11’1.7’’S— 65˚48’40.7’’W), Co´rdoba (31˚21’14.26’’S—64˚06’7.49’’´W), Corrientes (27˚21’59.07’’S—58˚ 16’58.9’’W), Jujuy (24˚29’11.51"S—64˚58’5.50"W) and La Rioja (30˚20’45.4"S—66˚19’33.4"W) Provinces of Argentina (Fig 1). Capture of specimens and their transport to the laboratory for subsequent identification were permitted by Secretarı´a de Ambiente (La Rioja Province),
Fig 1. Collection locations in Argentina. A: Puesto Viejo (Jujuy Province); B: National Route 12 (Corrientes Province); C: La Puerta (Catamarca Province); D: Chamical (La Rioja Province); E: Co´rdoba (Co´rdoba Province). doi:10.1371/journal.pone.0173052.g001
PLOS ONE | DOI:10.1371/journal.pone.0173052 February 24, 2017
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Mosquito molecular identification
Secretarı´a de Estado del Ambiente y Desarrollo Sustentable (Catamarca Province), Secretarı´a de Ambiente y Cambio Clima´tico (Co´rdoba Province), Ministerio de Ambiente (Jujuy Province) and Secretarı´a de ambiente y Desarrollo Sustentable (Corrientes Province). Field studies did not involve endangered or protected species. Larvae were individually reared and 17 males of each species were identified based on features of their genitalia [11] (S1 Table). Five sequences of C. bidens from Co´rdoba (Cba1507, Cba1514, Cba1526, Cba1532 and Cba1547) and one of C. interfor (Cba1505) corresponded to specimens which showed intermediate features of the male genitalia, similar to those described in Laurito and Almiro´n [13]. Fourth-instar larval and pupal exuviae and mounted male genitalia stored as vouchers are deposited in the Entomological Collection of the Centro de Investigaciones Entomolo´gicas de Co´rdoba (Universidad Nacional de Co´rdoba).
Molecular procedures Genomic DNA from each specimen was obtained from one or two legs preserved in absolute alcohol, using 60 μL of Chelex-100 5% (w/v) and 30 μL of NaCl 0.9% (w/v). The extract solution was vortexed and incubated at 100˚C for 15 min. After centrifugation at 13,000 rpm for 10 min at room temperature, 30–50 μL of supernatant was recovered. The DNA concentration was determined by comparison with known amounts of electrophoretic standards (λ/Hind III, Promega, USA), run on 0.8% agarose gels with ethidium bromide. DNA was eluted to a final concentration of 10 ng/μl and stored at -20˚C. ND4 (~336 bp) and COI (~658 bp) genes were amplified as in Da Costa-da-Silva et al. [29] and Folmer et al. [30], respectively, with minor modifications. The PCR products were electrophoresed in 1% agarose gels-TBE buffer and stained with ethidium bromide. All sequencing reactions were carried out in both directions using an ABI3730XL automatic sequencer (Macrogen Inc., Korea) with the same set of PCR primers. A panel of 12 microsatellite loci developed for other Culex (Culex) species (C. pipiens L. and C. quinquefasciatus Say) were screened, but only three were successfully amplified for C. bidens and C. interfor. The PCR reactions were performed as described in Fonseca et al. [31] using primers CQ41 and CQ11, and in Keyghobadi et al. [32] using primer GT51. The PCR products were visualized on 8% polyacrylamide gels stained with silver nitrate [33] and sized by comparison with a marker of 10 pb (Invitrogen, Argentina), using electrophoresis.
Mitochondrial and microsatellite data analyses The sequences for each mitochondrial gene were edited using BioEdit v. 7.2 [34]; primer regions were removed. Comparisons with available sequences using Basic Local Alignment Search Tool (blast.ncbi.nlm.nih.gov/Blast.cgi) were performed to check for sequence homology. Sequences were aligned by nucleotides using the Muscle algorithm [35] in SeaView v. 4 [36] and by amino acids using TranslatorX [37]. Aligned nucleotide sequences of the ND4 and COI genes were concatenated in a single data matrix using Mesquite [38]. Haplotype (Hd) and nucleotide (π) diversities for each gene and species were calculated using DnaSP v5 [39]. Allele frequencies at each microsatellite locus were calculated only for populations of Co´rdoba and La Rioja since very low sample size of both species was obtained in Catamarca; in Jujuy and Corrientes, only one of the species, C. bidens and C. interfor respectively, was collected (S1 Table).
Phylogenetic inference The best partitioning scheme and corresponding nucleotide substitution models were selected with the program PartitionFinder v. 1.0.1 [40], using the Bayesian Information Criterion
PLOS ONE | DOI:10.1371/journal.pone.0173052 February 24, 2017
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Mosquito molecular identification
(BIC). Bayesian phylogenetic (BP) analysis was conducted in MrBayes v. 3.1.2 [41] for the combined data matrix. Six independent runs of simultaneous MCMC (Markov Chain Monte Carlo) chains (temperature = 0.15) were performed for 2.5 million generations. Trees were sampled every 2,000 generations with the first 250 trees discarded as burn-in. The standard deviation of the split frequencies between runs (
Molecular identification of two Culex (Culex) species of the neotropical region (Diptera: Culicidae) Magdalena Laurito1☯*, Ana M. Ayala2☯, Walter R. Almiro´n1‡, Cristina N. Gardenal2‡ 1 Instituto de Investigaciones Biolo´gicas y Tecnolo´gicas (IIByT) CONICET-UNC. Centro de Investigaciones Entomolo´gicas de Co´rdoba (CIEC)—Universidad Nacional de Co´rdoba, Co´rdoba, Argentina, 2 Instituto de Diversidad y Ecologı´a Animal (IDEA) CONICET -Universidad Nacional de Co´rdoba, Co´rdoba, Argentina ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * [email protected]
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OPEN ACCESS Citation: Laurito M, Ayala AM, Almiro´n WR, Gardenal CN (2017) Molecular identification of two Culex (Culex) species of the neotropical region (Diptera: Culicidae). PLoS ONE 12(2): e0173052. doi:10.1371/journal.pone.0173052 Editor: Bi-Song Yue, Sichuan University, CHINA Received: December 13, 2016 Accepted: February 14, 2017 Published: February 24, 2017 Copyright: © 2017 Laurito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All sequences have been submitted to GenBank and the accession numbers are within the paper.
Abstract Culex bidens and C. interfor, implicated in arbovirus transmission in Argentina, are sister species, only distinguishable by feature of the male genitalia; however, intermediate specimens of the species in sympatry have been found. Fourth-instar larvae and females of both species share apomorphic features, and this lack of clear distinction creates problems for specific identification. Geometric morphometric traits of these life stages also do not distinguish the species. The aim of the present study was to assess the taxonomic status of C. bidens and C. interfor using two mitochondrial genes and to determine the degree of their reproductive isolation using microsatellite loci. Sequences of the ND4 and COI genes were concatenated in a matrix of 993 nucleotides and used for phylogenetic and distance analyses. Bayesian and maximum parsimony inferences showed a well resolved and supported topology, enclosing sequences of individuals of C. bidens (0.83 BPP, 73 BSV) and C. interfor (0.98 BPP, 97 BSV) in a strong sister relationship. The mean K2P distance within C. bidens and C. interfor was 0.3% and 0.2%, respectively, and the interspecific variation was 2.3%. Bayesian clustering also showed two distinct mitochondrial lineages. All sequenced mosquitoes were successfully identified in accordance with the best close match algorithm. The low genetic distance values obtained indicate that the species diverged quite recently. Most morphologically intermediate specimens of C. bidens from Co´rdoba were heterozygous for the microsatellite locus GT51; the significant heterozygote excess observed suggests incomplete reproductive isolation. However, C. bidens and C. interfor should be considered good species: the ventral arm of the phallosome of the male genitalia and the ND4 and COI sequences are diagnostic characters.
Funding: PICT 2013-1254 http://www.agencia. mincyt.gob.ar/ Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica. ML.
Introduction
Competing interests: The authors have declared that no competing interests exist.
The genus Culex L., with a worldwide distribution and 770 species grouped in 26 subgenera [1], is widely known for its medical and veterinary importance. Among the subgenera,
PLOS ONE | DOI:10.1371/journal.pone.0173052 February 24, 2017
1 / 12
Mosquito molecular identification
subgenus Culex includes 200 species [1], many of them recognized as vectors of filarial nematodes [2] and several arboviruses [3]. The subgenus is not monophyletic [4,5,6] and the infrasubgeneric categories are based on superficial similarities that may not reflect natural relationships [7]. The subgenus exhibits polymorphic features and exceptional forms [8] and is arranged in an informal classification that includes Series, Groups, Subgroups and Complexes, with nine species without placement [1]. Morphological identification of Culex (Culex) species is difficult because anatomical traits of fourth-instar larvae and females are polymorphic and overlap among species. Only morphological features of the male genitalia provide a wealth of taxonomic characters that allow reliable identification of the species, as well as synapomorphic traits that contribute to revolving phylogenetic relationships in the subgenus [9]. Culex bidens Dyar and C. interfor Dyar are a clear example of the problematic identification described above. The former is widely distributed in many countries of the Neotropical Region from Mexico to Argentina; the latter is restricted to Argentina and Paraguay [10]. The species are grouped in a well-supported clade as sister species on the basis of morphological characters [9]. These species share apomorphic features of fourth-instar larvae and females, being only distinguishable by some male genitalia traits [9]. The two species share features of the subapical lobe of the gonocoxopodites, paraproct and dorsal arms of the phallosome. Culex bidens differs by having lateral plate with 1–3 (usually 2) large dorsolaterally directed teeth, 0–3 minute conical denticles and the ventral arm developed as a spine bent dorsolaterally. The lateral plate of C. interfor has a single strong tooth directed dorsolaterally (infrequently with denticles), and the ventral arm is small, triangular and laterally directed flap-like process [11]. Besides morphological characters, geometric morphometric procedures were also assessed to distinguish these species from each other and from other species [12], revealing that shapes of wings of adults and dorsomentum of larvae could not distinguish them. Furthermore, intermediate features in male genitalia in sympatric populations of the two species have recently been found, suggesting that successful interbreeding could be occurring in the localities of Resistencia (Chaco Province), Co´rdoba and Rı´o Seco (Co´rdoba Province), Puerto Iguazu´ (Misiones Province) and Chamical (La Rioja Province) of Argentina [13]. Distinguishing closely related species using morphological traits is not easy, even more so if there are intermediate specimens. Accurate identification is of paramount importance to determine areas of potential risk of disease transmission, particularly when dealing with invasive mosquito species that are potential disease vectors. The need to distinguish C. bidens and C. interfor is important because they have been implicated in the transmission of two arboviruses. Culex bidens was assumed to be the vector of Venezuelan Equine Encephalitis virus (VEEV) during the epizootic in Chaco Province in 1988 [14] and C. interfor is considered a secondary vector of Saint Louis Encephalitis virus (SLEV) [15] in Co´rdoba, Argentina. To achieve a correct identification of these species, alternative tools, such as molecular methods, are needed. To be effective, a diagnostic molecular marker must demonstrate consistent differences between closely related species [16]. Mitochondrial genes are considered good markers because of their abundance, lack of introns, limited exposure to recombination, and haploid mode of inheritance [17]. Among mitochondrial genes, a fragment of the cytochrome c oxidase subunit I (COI) has been largely employed for taxon barcoding [18], and to estimate genetic divergence among phylogenetically close species [19]. In mosquitoes, COI barcode sequences have been used to successfully identify species [20,21,22] and to recognize species complexes [23,24,25]. Even though Laurito et al. [26] revealed that the COI barcode fragment does not contain enough information to identify all species of Culex (Culex) from the Neotropical Region, about 40% of the samples clustered with their conspecifics in that study. Another DNA region, the nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4) has
PLOS ONE | DOI:10.1371/journal.pone.0173052 February 24, 2017
2 / 12
Mosquito molecular identification
recently been employed for specific identification in Aedes koreicus (Edwards) populations far away from its known natural range of distribution [27]. Cameron et al. [28] designed a molecular assay based on ND4 sequences to identify species of Aedes Meigen. The lack of appropriate diagnostic morphological characters to identify C. bidens and C. interfor in all stages except males, their close phylogenetic relationship, the sympatric occurrence of intermediate specimens and the poor differentiation based on geometric morphometrics lead us to question the taxonomic rank of these species. The aim of the present study was to assess the taxonomic status of C. bidens and C. interfor using a mitochondrial DNAbased phylogenetic molecular approach. Additionally, three microsatellite loci were tested to get complementary information based on other than maternally inherited markers, which could provide evidence about the degree of reproductive isolation between these species.
Material and methods Mosquito collection Fourth-instar larvae were collected between 2012 and 2015 in the Catamarca (28˚11’1.7’’S— 65˚48’40.7’’W), Co´rdoba (31˚21’14.26’’S—64˚06’7.49’’´W), Corrientes (27˚21’59.07’’S—58˚ 16’58.9’’W), Jujuy (24˚29’11.51"S—64˚58’5.50"W) and La Rioja (30˚20’45.4"S—66˚19’33.4"W) Provinces of Argentina (Fig 1). Capture of specimens and their transport to the laboratory for subsequent identification were permitted by Secretarı´a de Ambiente (La Rioja Province),
Fig 1. Collection locations in Argentina. A: Puesto Viejo (Jujuy Province); B: National Route 12 (Corrientes Province); C: La Puerta (Catamarca Province); D: Chamical (La Rioja Province); E: Co´rdoba (Co´rdoba Province). doi:10.1371/journal.pone.0173052.g001
PLOS ONE | DOI:10.1371/journal.pone.0173052 February 24, 2017
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Mosquito molecular identification
Secretarı´a de Estado del Ambiente y Desarrollo Sustentable (Catamarca Province), Secretarı´a de Ambiente y Cambio Clima´tico (Co´rdoba Province), Ministerio de Ambiente (Jujuy Province) and Secretarı´a de ambiente y Desarrollo Sustentable (Corrientes Province). Field studies did not involve endangered or protected species. Larvae were individually reared and 17 males of each species were identified based on features of their genitalia [11] (S1 Table). Five sequences of C. bidens from Co´rdoba (Cba1507, Cba1514, Cba1526, Cba1532 and Cba1547) and one of C. interfor (Cba1505) corresponded to specimens which showed intermediate features of the male genitalia, similar to those described in Laurito and Almiro´n [13]. Fourth-instar larval and pupal exuviae and mounted male genitalia stored as vouchers are deposited in the Entomological Collection of the Centro de Investigaciones Entomolo´gicas de Co´rdoba (Universidad Nacional de Co´rdoba).
Molecular procedures Genomic DNA from each specimen was obtained from one or two legs preserved in absolute alcohol, using 60 μL of Chelex-100 5% (w/v) and 30 μL of NaCl 0.9% (w/v). The extract solution was vortexed and incubated at 100˚C for 15 min. After centrifugation at 13,000 rpm for 10 min at room temperature, 30–50 μL of supernatant was recovered. The DNA concentration was determined by comparison with known amounts of electrophoretic standards (λ/Hind III, Promega, USA), run on 0.8% agarose gels with ethidium bromide. DNA was eluted to a final concentration of 10 ng/μl and stored at -20˚C. ND4 (~336 bp) and COI (~658 bp) genes were amplified as in Da Costa-da-Silva et al. [29] and Folmer et al. [30], respectively, with minor modifications. The PCR products were electrophoresed in 1% agarose gels-TBE buffer and stained with ethidium bromide. All sequencing reactions were carried out in both directions using an ABI3730XL automatic sequencer (Macrogen Inc., Korea) with the same set of PCR primers. A panel of 12 microsatellite loci developed for other Culex (Culex) species (C. pipiens L. and C. quinquefasciatus Say) were screened, but only three were successfully amplified for C. bidens and C. interfor. The PCR reactions were performed as described in Fonseca et al. [31] using primers CQ41 and CQ11, and in Keyghobadi et al. [32] using primer GT51. The PCR products were visualized on 8% polyacrylamide gels stained with silver nitrate [33] and sized by comparison with a marker of 10 pb (Invitrogen, Argentina), using electrophoresis.
Mitochondrial and microsatellite data analyses The sequences for each mitochondrial gene were edited using BioEdit v. 7.2 [34]; primer regions were removed. Comparisons with available sequences using Basic Local Alignment Search Tool (blast.ncbi.nlm.nih.gov/Blast.cgi) were performed to check for sequence homology. Sequences were aligned by nucleotides using the Muscle algorithm [35] in SeaView v. 4 [36] and by amino acids using TranslatorX [37]. Aligned nucleotide sequences of the ND4 and COI genes were concatenated in a single data matrix using Mesquite [38]. Haplotype (Hd) and nucleotide (π) diversities for each gene and species were calculated using DnaSP v5 [39]. Allele frequencies at each microsatellite locus were calculated only for populations of Co´rdoba and La Rioja since very low sample size of both species was obtained in Catamarca; in Jujuy and Corrientes, only one of the species, C. bidens and C. interfor respectively, was collected (S1 Table).
Phylogenetic inference The best partitioning scheme and corresponding nucleotide substitution models were selected with the program PartitionFinder v. 1.0.1 [40], using the Bayesian Information Criterion
PLOS ONE | DOI:10.1371/journal.pone.0173052 February 24, 2017
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Mosquito molecular identification
(BIC). Bayesian phylogenetic (BP) analysis was conducted in MrBayes v. 3.1.2 [41] for the combined data matrix. Six independent runs of simultaneous MCMC (Markov Chain Monte Carlo) chains (temperature = 0.15) were performed for 2.5 million generations. Trees were sampled every 2,000 generations with the first 250 trees discarded as burn-in. The standard deviation of the split frequencies between runs (
